Abstract Dysregulation of absorption of dietary triglycerides (TGs) leads to adiposity or lipodystrophy, each of which can cause insulin insensitivity. Autophagy degrades unwanted cytoplasmic contents in lysosomes to maintain quality control. We have shown that autophagy degrades cytosolic lipid droplets (LDs) in multiple tissues via lipophagy. Whether autophagy in gut contributes to absorption of dietary TGs remains unknown. Dietary triglycerides (TGs) are absorbed by enterocytes as free fatty acids, which are re-esterified to TGs at the endoplasmic reticulum (ER) membrane by distinct TG synthesizing enzymes. Nascently produced TGs enter the ER lumen to form chylomicrons for secretion or are stored transiently as cytosolic LDs. In this application, using cultured enterocytes and mice as models, we will investigate how interplay between the nutrient sensor mTORC1, autophagy protein LC3, and TG synthesis enzymes regulates the fate of ingested lipid. We hypothesize that free fatty acid availability in enterocytes activates and localizes mTOR to ER membranes where it phosphorylates local pools of LC3. Phosphorylated (P)-LC3 is uncoupled from canonical autophagy and serves as scaffolds that interact with TG synthesis enzymes to regulate TG biogenesis ? a key step driving chylomicron formation and secretion. We propose further that in the physiological state lipophagy contributes to clearance of transiently-stored cytosolic LDs, thus limiting the amount of TGs available for secretion. To test these hypotheses, we propose the following specific aims: S.A. 1: To characterize the phosphorylations and interactome of LC3 at the ER membrane. In this aim, we will identify the phosphorylation signature and interacting partners of LC3 at the ER membrane during lipid availability. We will determine the contribution of mTORC1 or its down-stream target ULK1 to LC3 phosphorylation. We will use site-directed mutagenesis to study the function of each of the identified phosphorylations towards TG synthesis in the ER, and TG secretion from the enterocyte. We will determine which of the newly-identified LC3 interacting partners regulate TG synthesis and secretion. S.A. 2: To dissect the interplay between mTOR, LC3, and lipophagy in dietary lipid absorption. In S.A. 2, we will use site-directed mutagenesis in cultured Caco2 intestinal cells, and novel mice models, to dissect the crosstalk between mTOR, LC3 and ER-localized TG synthesis enzymes in regulation of TG synthesis and TG entry into the ER. S.A. 3: To determine the contribution of gut mTOR signaling and lipophagy to development of metabolic syndrome during obesity. mTORC1 signaling and autophagy are each suppressed in obesity, which per se associates with increased absorption of dietary TGs. Consequently, we will use high fat feeding of mouse models of gain-of-function of mTOR signaling or loss-of- function of autophagy to explore the contribution of gut-specific mTOR and autophagy to development of metabolic syndrome. We will explore whether strategically-timed inhibition of mTOR signaling by rapamycin will reduce intestinal TG absorption and prevent metabolic syndrome. We will establish paradigm-shifting roles for mTOR, LC3, and autophagy in the molecular regulation of dietary TG absorption. Our findings will create a framework to consider how mTOR and LC3 communicate with novel interacting partners at the ER to regulate fundamental aspects of lipid metabolism. Significance: The metabolic syndrome is a significant global health problem affecting greater than 44% of the U.S. population aged more than 50 years. The metabolic syndrome affects health-span through effects on cardio/cerebrovascular health, locomotion, vision, cognition, and tumor development. The current proposal will delineate a novel crosstalk of mTOR and ATG protein in the regulation of gut TG metabolism, setting the basis for therapeutic modulation of mTOR signaling in the gut to prevent or treat the metabolic syndrome.